In developed countries, stroke is the third leading cause of death and the primary cause of acquired physical or cognitive impairment. Vascular dementia is the second leading cause of dementia, after Alzheimer's disease. CADASIL (for cerebral autosomal dominant arteriopathy with lubcortical infarcts and leukoencephalopathy) causes a type of stroke and dementia whose key features include recurrent subcortical ischaemic events, progressive vascular dementia, craniofacial paralysis, migraine and mood disorders with severe depression (Chabriat, H. et al., (1995) Lancet 346: 934-939). These symptoms usually start to appear at about 45 years of age, and patients typically die by 65. The condition is believed to be largely undiagnosed and therefore the prevalence is not precisely known.
CADASIL is associated with diffuse white-matter abnormalities on neuroimaging (Tournier-Lasserve, E. et. al., (1991) Stroke 22:1297-1302). Pathological examination reveals multiple small, deep cerebral infarcts, a leukoencephalopathy and a non-atherosclerotic, non-amyloid angiopathy involving mainly the small cerebral arteries.(Baudrimont, M. et al., (1993) Stroke 24: 122-125). Severe alterations of vascular smooth muscle cells are evident on ultrastructural analysis (Ruchoux, M. M. et al., (1995) Acta. Neuropathol. 89:500-512).
It has recently been shown that the human Notch3 gene, located on chromosome 19, is mutated in CADASIL patients (Joutel. A. et al., (1996) Nature 383: 707-710). Most of the mutations cause amino acid changes in the extracellular domain. Therefore, disruption of the Notch signaling pathway appears to be responsible for CADASIL stroke and dementia.
Defects in the Notch signaling pathway may also be involved in other neurological diseases, e.g., Alzheimer's disease. In fact, approximately 10% of cases of Alzheimer's disease are associated with mutations in genes encoding the amyloid precursor proteins, presenilin 1 (PS1) and presenilin 2 (PS2). About 25% of early-onset familial Alzheimer's cases are associated with a mutation in PS1. PS1 and PS2 are transmembrane proteins which are homologous to the C. elegans protein encoded by the sel-12 gene, which has been shown to be genetically linked to the C. elegans lin-12 gene, which encodes a Notch-family receptor (Levitan, D. and I. Greenwald (1995) Nature 377:351-354; PS1 and PS2 are further described in PCT Application WO 96/34099; Sel12 is further described in PCT Application WO 97/11956). Furthermore, targeted disruption of PS1 in mice results in reduced expression of mRNA encoding Notch1 and Delta-like gene 1 (Dll1), a vertebrate Notch ligand, in the presomitic mesoderm compared to control mice (Wong et al. (1997) Nature 387:288). This indicates that PS1 is required for the spatiotemporal expression of genes involved in the Notch signaling pathway.
The Notch signaling pathway comprises Notch proteins, which are membrane proteins, and proteins interacting with Notch proteins, termed Delta proteins. The product of the Delta gene, acting as a ligand, and that of the Notch gene, acting as a receptor, are key components in a lateral-inhibition signaling pathway that regulates the detailed patterning of many different tissues in Drosophila (Vassin, H., et al., (1987) EMBO J 6:3431-3440; Kopczynski, C. et al., (1988) Genes Dev. 2:1723-1735; Fehon, R. G. et al., (1990) Cell 61:523-534; Artavanis-Tsakonas, S. et., al., (1991) Trends, Genet. Sci. 7:403-407; Heitzler, P. et. al., (1991) Cell 64: 1083-1092; Greenwald, I. et al., (1992) Cell 68: 271-281; Fortini, M et al., (1993) Cell 75: 124501247; and Muskavitch, M. (1994) Devl. Biol. 166:415-430). During neurogenesis in particular, neural precursors, by expressing Delta, inhibit neighboring Notch-expressing cells from becoming committed to a neural fate. Mutations leading to a failure of lateral inhibition cause an overproduction of neurons, giving rise to a phenotype termed the "neurogenic phenotype" in Drosophila. For example, loss of Notch1 leads to somite defects and embryonic death in mice, whereas constitutively active mutant forms of Notch1 appear to inhibit cell differentiation in Xenopus and in cultured mammalian cells (Swiatek et al. (1994) Genes Dev. 8:707; Conlon et al. (1995) J. Development 121:1533; Lopan et al. (1994) Development 120:2385; and Nye et al. (1994) Development 120:2421). Similarly, reduced activity of X-Delta1 causes more cells to become primary neurons, whereas raised activity causes fewer cells to become primary neurons (Chitnis et al. (1995) Nature 375:761). Furthermore, loss of Dll1 function in mice leads to excessive neuronal differentiation, resulting in severe patterning defects in the paraxial mesoderm and a hyperplastic central nervous system (CNS) (Hrabe de Angelis et al. (1997) Nature 386:717). Thus, the Notch signaling pathway, in particular Delta proteins, mediate lateral inhibition during neurogenesis so that only a limited proportion of cells having the potential to become neurons will in fact differentiate into neurons.
The Notch family of proteins are transmembrane receptors containing several conserved peptide motifs. The extracellular domains contain many tandemly repeated copies of an epidermal growth factor (EGF) like motif. The intracellular domains contain six copies of another conserved motif, termed the Cdc10/ankyrin repeat. Both the EGF and the ankyrin-repeat motifs are found in many different proteins and, in at least some cases, they have been shown to be involved in protein-protein interactions.
The Drosophila Notch protein encodes a glycosylated transmembrane receptor having a molecular mass 350 KD, which is involved in cell-fate specification during development (Wharton, K. A. et al., (1985) Cell 43:567-581); Artavanis-Tsakonas, S. et al., (1995) Science 268: 225-232). Based on analysis of Drosophila mutants, it is thought that Notch is a receptor having different functional domains, with the intracellular domain having the intrinsic signal-transducing activity of the intact protein and the extracellular domain a regulating activity (Rebay, I et al., (1993) Cell 74: 319-329).
Several Notch homologues have been identified in vertebrates (Larsson, C., et al., (1994) Genomics 24: 253-258). Three Notch proteins (Notch1, also called TAN1, Notch2, and Notch3) have been characterized in humans (Ellisen, L. W. et al., (1991) Cell 66:649-661; Stifani, S. et al., (1992) Nature Genet. 2: 119-127). Notch1 gene translocations have been associated with a minority of T-cell lymphoblastic leukemias (Aster, J. (1994) Cold Spring Harb. Symp. Quant. Biol. 59:125-136) and Notch3 has been linked with CADASIL.
A protein interacting with Notch was first discovered in Drosophila and has been called Delta protein. This protein encodes a transmembrane protein ligand, which contains tandem arrays of epidermal growth factor-like repeats in the extracellular domain. The Delta and Notch proteins can directly bind to each other and specific EGF-like repeats are sufficient and necessary for this binding (Fehon, R. G. et al., (1990) Cell 61:523-534; Rebay I., et al., (1991) Cell 67:687-699; and Lieber, T., et al., (1992) Neuron 9: 847-859).
In addition to the Drosophila Delta protein, a chick Delta homologue, C-Delta protein (Henrique, D et al., (1995) Nature 375: 787-790 and GenBank Accession No. U26590) two Xenopus homologues, X-Delta-1 and X-Delta-2 (Chitnis, A. et al., (1995) Nature 375:761-766 and GenBank Accession Nos. L42229 and U70843), a mouse homologue (GenBank Accession No. X80903), a delta-like human gene 1(Dlk) (Bettenhausen, B. et al., (1995) Development 121:2407-2418) a rat homologue (GenBank Accession No. U78889), and a Zebrafish homologue (GenBank Accession No. Y11760) have been identified. Xenopus, chick and mouse Delta genes are also disclosed in International Patent Application No. PCT/US96/11178 (Publication No. WO 97/01571. The patent application also discloses a partial and error prone human Delta homolog (hD1). Nucleotide sequence of human Notch genes are disclosed in International Patent Application No. PCT/US92/03651 (Publication No. WO 92/19734) and International Patent Application No. PCT/US93/09338 (Publication No. WO 94/07474).
Notch signaling pathway therapeutics, in particular Delta therapeutics are highly desirable for treating various diseases and disorders, including neurological and vascular disorders.